PROJECT SUMMARY Herpes simplex virus 1 (HSV-1) is a widespread human pathogen that uses latency to remain for life in peripheral nerve ganglia. Reactivation of latent HSV-1 is responsible for a spectrum of diseases but there currently are no effective therapies to eliminate the latent reservoir or prevent reactivation. After decades of study, our understanding of the molecular basis of latency/reactivation and associated pathogenesis remains incomplete. Most studies use live-rodent infection models but these are difficult to manipulate and do not fully recapitulate the properties of HSV-1 infection in humans. Use of primary neuron culture models as an alternative to animals has gained traction in the last ten years, and has provided many important insights into the contribution of host factors that are now guiding further research in animals. The field has matured to a point where we need models using human neurons. This proposal capitalizes on our success in developing a new model of HSV-1 latency in neurons differentiated in vitro from an NIH-approved human embryonic stem cell line. We will use this model to test two important hypotheses that have been very difficult to explore in rodents or rodent-derived neurons. Aim 1 will investigate the impact of preventing expression of the latency- associated non-coding RNAs (LATs) and latency-specific microRNAs. These are expressed at high levels in infected human ganglia but it has been extremely difficult to elucidate their biological roles in rodent models. Aim 2 will reevaluate the contribution of the viral transactivator VP16 to latency and reactivation in the context of human neurons. VP16 is recruited to viral immediate-early (IE) promoters through interaction with the cellular DNA-binding protein Oct-1 (POU2F1). The amino acid sequence of Oct-1 is highly conserved in vertebrates but deviates noticeably in rodents. Substitutions at just two positions on the surface of the rodent Oct-1 POU homeodomain reduces the binding of VP16 to Oct-1 by 100-fold, with an equivalent reduction in VP16-dependent transactivation. To understand the biological consequences of this striking but largely ignored difference, we will use CRISPR/Cas9 technology to exchange just three residues in human Oct-1 in the stem cells to the mouse equivalents (E30D/M33L/D36E), differentiate these into neurons and measure both the efficiency of latency establishment and levels of spontaneous reactivation, a feature of human infections that is missing in murine models. This will test the idea that mismatches in specific molecular interactions can significantly alter the dynamics of the virus-host relationship. Together these studies will provide strong impetus to use in vitro models based on human neurons and develop of improved mouse models that more fully recapitulate the behavior of HSV-1 in it's human host.